The Splenic Microenvironment Is a Novel Site of Relapse Following ABT-199 Treatment

T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy arising from the transformation of immune T-cell lymphocytes. Early T-cell progenitor (ETP-ALL) is a subgroup particularly associated with a poor prognosis and a high risk for relapse. While the leukaemia initially develops in the thymus it spreads in the blood to the bone marrow, lymph nodes and often the spleen. Interestingly, splenomegaly was previously associated with a poor prognosis in leukemic patients. Recently, it was shown that ETP-ALL is dependent on the expression of the anti-apoptotic protein BCL-2, and is sensitive to inhibition with ABT-199, a BCL-2 specific BH3 mimetic. However, one issue with targeted agents, like ABT-199, is the development of resistance to treatment. Our aim was to determine potential in vivosites of resistance/relapse following ABT-199 treatment using a xenograft model of ETP-ALL. We confirmed that the ETP-ALL LOUCY cell line is BCL-2 dependent and then labelled it with luciferase to enable visualisation of the leukaemia in vivo. Following establishment of the leukaemia in NOD/SCID gamma mice, as assessed by hCD45+, the mice were randomised to receive vehicle control or 50 mg/kg ABT-199 by oral gavage daily for two weeks. While the mice were initially sensitive to ABT-199, the leukaemia started to progress while on treatment. Interestingly, there appeared to be a selective redistribution of the leukaemia to the spleen following ABT-199 treatment. Indeed, LOUCY cells isolated from the spleen of the mice had a reduced BCL-2 dependence, as assessed by BH3 profiling. The reduced BCL-2 dependence correlated with reduced BCL-2 expression at both the mRNA and protein level. Next, we confirmed that human splenic fibroblasts (HSF) co-cultured with the LOUCY cell line in vitro also altered BCL-2 dependence and expression using BH3 profiling and Western blotting. To identify potential splenic cytokines involved in the regulation of BCL-2 protein expression in ETP-ALL we performed a screening cytokine array. Upon co-culture of the LOUCY cells with HSF there was an increased expression of IL-6, this was confirmed using ELISA. Using an IL-6 receptor antibody we confirmed that blocking IL-6 receptor reversed the change in BCL-2 dependence in the presence of the splenic microenvironment. Lastly, we confirmed in a T-ALL patient-derived xenograft, that is BCL-2 dependent, that the splenic microenvironment alters the mitochondrial apoptotic threshold. Currently, there are reports in the literature of ETP-ALL patients being treated with ABT-199. While there have been numerous studies lately describing cell autonomous events leading to ABT-199 resistance, our novel finding that the splenic microenvironment is a site of relapse is potentially of great clinical importance for BCL-2 dependent leukemia's. Disclosures Ni Chonghaile: AbbVie: Research Funding.

Download Full-text

Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Blood ◽

10.1182/blood-2020-140060 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 21-21

Author(s):

Gisele Olinto Libanio Rodrigues ◽

Julie Hixon ◽

Hila Winer ◽

Erica Matich ◽

Caroline Andrews ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cell Line ◽

Lymphoblastic Leukemia ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Cell Counts

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

Download Full-text

Silencing VEGF enhances the radiosensitivity of the radioresistant human nasopharyngeal carcinoma cell line CNE-2R by inhibiting autophagy through the activation of mTOR pathway

10.21203/rs.3.rs-26678/v1 ◽

2020 ◽

Author(s):

Li Chen ◽

Guoxiang Lin ◽

Kaihua Chen ◽

Fangzhu Wan ◽

Yongchu Sun ◽

...

Keyword(s):

Dna Damage ◽

Nasopharyngeal Carcinoma ◽

Cell Line ◽

Western Blotting ◽

Xenograft Model ◽

Mtor Pathway ◽

Angiogenic Factor ◽

Cell Counting

Abstract Background: Vascular endothelial growth factor (VEGF) is an important pro-angiogenic factor. VEGF was reported to promote the occurrence of autophagy, which enhanced to the radioresistance of tumors. The purpose of our study was to investigate the influence of VEGF silencing on the radiosensitivity of nasopharyngeal carcinoma radioresistant cell line CNE-2R and the underlying mechanisms.Methods: The radiosensitivity of CNE-2R cells after silencing VEGF was detected by cell counting kit 8 (CCK-8) and clonogenic assay, cell cycle and apoptosis was subjected to flow cytometry. DNA damage and autophagy were observed by immunofluorescence and western blotting. The interaction between VEGF and mTOR was confirmed by western blotting and co-immunoprecipitation analysis. In vivo, the effect of VEGF on radiosensitivity of NPC cells was investigated through xenograft model, furthermore, immunohistochemistry and TUNEL assay were used to further verify the relationship between autophagy and radiosensitivity in NPC after VEGF depletion.Results: Downregulation of VEGF significantly inhibited cell proliferation and induced apoptosis of CNE-2R cells after radiotherapy in vitro and in vivo. In addition, VEGF knockdown not only decreased autophagy level, but also delayed the DNA damage repair in CNE-2R cells after irradiation. Mechanistically, silencing VEGF suppressed autophagy through the activation of mTOR pathway.Conclusion: VEGF depletion increased radiosensitivity of NPC radioresistant cell CNE-2R by suppressing autophagy via the activation of mTOR pathway.

Download Full-text

A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo

Leukemia ◽

10.1038/leu.2016.388 ◽

2016 ◽

Vol 31 (8) ◽

pp. 1743-1751 ◽

Cited By ~ 86

Author(s):

S Hipp ◽

Y-T Tai ◽

D Blanset ◽

P Deegen ◽

J Wahl ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cell ◽

Plasma Cells ◽

Ex Vivo ◽

Xenograft Model ◽

Selective Lysis ◽

Bispecific T Cell Engager

Abstract B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein that is expressed on malignant plasma cells of multiple myeloma (MM) patients and therefore is an ideal target for T-cell redirecting therapies. We developed a bispecific T-cell engager (BiTE) targeting BCMA and CD3ɛ (BI 836909) and studied its therapeutic impacts on MM. BI 836909 induced selective lysis of BCMA-positive MM cells, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA-negative cells were not affected. Activity of BI 836909 was not influenced by the presence of bone marrow stromal cells, soluble BCMA or a proliferation-inducing ligand (APRIL). In ex vivo assays, BI 836909 induced potent autologous MM cell lysis in both, newly diagnosed and relapsed/refractory patient samples. In mouse xenograft studies, BI 836909 induced tumor cell depletion in a subcutaneous NCI-H929 xenograft model and prolonged survival in an orthotopic L-363 xenograft model. In a cynomolgus monkey study, administration of BI 836909 led to depletion of BCMA-positive plasma cells in the bone marrow. Taken together, these results show that BI 836909 is a highly potent and efficacious approach to selectively deplete BCMA-positive MM cells and represents a novel immunotherapeutic for the treatment of MM.

Download Full-text

Induction of acetylcholine receptor-specific suppression. An in vitro model of antigen-specific immunosuppression in myasthenia gravis.

Journal of Experimental Medicine ◽

10.1084/jem.159.1.292 ◽

1984 ◽

Vol 159 (1) ◽

pp. 292-304 ◽

Cited By ~ 11

Author(s):

S Bogen ◽

E Mozes ◽

S Fuchs

Keyword(s):

T Cell ◽

Myasthenia Gravis ◽

Clinical Importance ◽

Lymphocyte Transformation ◽

Specific Suppression ◽

Target Autoantigen ◽

High Concentrations ◽

In Vitro Induction

This report describes the in vivo and in vitro induction of murine (AChR)-specific suppressor T cells (Ts) and T cell factors (TsF), and the development of an appropriate assay system for their measurement. The assay described is based on the in vitro Mishell-Dutton culture system. Using this assay, it was shown that the AChR-specific helper cell is an Lyt-2- radiosensitive T cell. Moreover, the proliferating cell measured in the lymphocyte transformation assay was shown to provide AChR-specific T cell help. In vivo induction of Ts cells is achieved by injection of soluble AChR; potent AChR-specific suppression is found in the spleen 1 wk later. In vitro induction of Ts cells involves the primary education of naive splenocytes by culturing them with high concentrations of AChR. Both the in vivo- and in vitro-induced Ts cells were shown to secrete AChR-specific factors that mediate their suppressive effects. The possibility of specifically suppressing the AChR-immune response may be of a particular clinical importance since the AChR is the target autoantigen in the neuromuscular autoimmune disease myasthenia gravis.

Download Full-text

Ectonucleosidase CD39 Is Highly Expressed on ATLL Cells and Suppresses the Immune Response through the Adenosine Pathway

Blood ◽

10.1182/blood-2018-99-118555 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3720-3720

Author(s):

Yasuhiro Nagate ◽

Sachiko Ezoe ◽

Jiro Fujita ◽

Takafumi Yokota ◽

Michiko Ichii ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Line ◽

Tumor Cells ◽

Research Funding ◽

Poly I:C ◽

Acute Type ◽

Atll Cell

Abstract Background: Adult T-cell leukemia/lymphoma (ATLL) is a mature T-cell neoplasm, linked to the human T-cell lymphotropic virus, HTLV-1. Patients with ATLL are often at the risk of opportunistic infections. Some studies suggested that ATLL cells originate from HTLV-1-infected regulatory T cells (Tregs). It could be possible that this immunocompromised state is caused by the function of ATLL cells having similar phenotypes with Tregs. In this study, we examined the expression of immunosuppressive molecules associated with Tregs in ATLL cells, and analyzed their roles in the function of ATLL cells. Methods: The protocol of this study was approved by the Investigational Review Board of Osaka University Hospital. Peripheral blood mononuclear cells (PBMCs) were collected from 10 asymptomatic HTLV-1 carriers and 22 ATLL patients (1 with smoldering type, 5 with chronic type, 2 with lymphoma type, and 14 with acute type) after getting informed consent. PBMCs from 3 ATLL patients were separated into CD4+ CD7- CADM1+ATLL cells and adjacent CD4+CD7+ CADM1-normal T cells using Fluorescence-activated Cell Sorter (FACS), and cells in each fraction were subjected to total RNA sequencing experiments. Based on the results, we examined the expression patterns of CD39 and CD73 in HTLV-1 carriers or each type of ATLL patients, and also analyzed the immune functions of these molecules in ATLL tumor cells. Results: We compared whole transcriptome of ATLL cells and normal CD4+cells. Bioinformatic analyses showed that many genes associated with immunosuppressive functions were elevated or downregulated in ATLL cells. Among these genes we focused on CD39, CD73 and CD26, because they have recently been reported to be strongly associated with the functions of Tregs. CD39, expressed on normal Tregs, and extrinsic CD73 have immunosuppressive potential by catalyzing adenosine from extracellular ATP, and CD26 has opposite potential by resolving adenosine, which have a strong anti-inflammatory function and plays major role in Treg-mediated immunosuppression. We found that all of 4 ATLL cell lines (MJ, MT1, MT2, MT4) expressed CD39, but not CD73 just as human effector Tregs. Tumor cells from 12 acute ATLL patients (86%) and 2 chronic ATLL patients (40%) expressed CD39, but the expressions of CD73 were various. Also in asymptomatic carriers, we could detect CD39 and/or CD73 positive in CD7- CADM1+ abnormal fraction of CD4+cells. On the other hand, CD26, normally expressed on human CD4+Th cells other than effector Tregs, was negative in ATLL cell lines and primary ATLL cells except for cells in abnormal fraction of one asymptomatic carrier. CD39 negative cases in chronic/smoldering type tended to show slower disease progression after the blood collection. Next, the role of CD39 and/or CD73 in ATLL cells was assessed in vitro and in vivo. As expected, CD39+ ATLL cells converted significantly more extracellular ATP than CD39- ATLL cells, and mass spectrometry analysis of AMP/adenosine concentration identified the AMPase activity of CD73+ ATLL cells. Furthermore, we established CD39 knockout (KO) cells from ATL cell-line MJ using CRISPR/Cas9 system, and performed in vitro suppression assays for assessment of immunosuppressive function. Although wild type MJ suppressed the growth of normal CD4+ and CD8+ T cells, KO MJ did little. Next, we analyzed the role of CD39 in the progression of tumor cells in vivo. We transplanted mouse T-cell lymphoma cell-line EG7-OVA artificially expressing CD39 or mock into mice subcutaneously. The coinjection of immunoadjuvant poly(I:C) significantly suppressed the tumor growth of mock cells, but the tumor sizes of CD39 expressing cells were almost the same as those of mock cells without poly(I:C) injection (Figure). Conclusion: In this study, we reported that most of ATLL cells in acute type patients express CD39+ CD26- just as Tregs, and that CD39- KO of ATLL cell line cancelled its immunosuppressive effects, and forcibly expressed CD39 on tumor cells rejected the anti-tumor immunity in vivo. From these data, we clarified the pathological mechanism of immunosuppressive function in ATLL cells, and also showed that CD39 expression could be used as a prognostic clue and be a new therapeutic target of ATLL. Disclosures Ezoe: TAIHO Phamaceutical Co., Ltd.: Research Funding. Yokota:Celgene: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer Inc.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; MSD K.K.: Research Funding. Ichii:Novartis Pharma K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Celgene K.K.: Speakers Bureau. Shibayama:Novartis Pharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Fujimoto Pharmaceutical: Honoraria, Research Funding; Jansen Pharmaceutical K.K: Honoraria; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding. Oritani:Novartis Pharma: Speakers Bureau. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

Download Full-text

Evaluation of Two Novel Therapeutics Against Human and Canine Osteosarcoma

10.21203/rs.3.rs-51104/v1 ◽

2020 ◽

Author(s):

Heather Wilson-Robles ◽

Tasha Miller ◽

Chao Sima ◽

Jianping Hua ◽

Milana Cypert ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Xenograft Model ◽

Western Blots ◽

Primary Bone Tumor ◽

Murine Xenograft Model ◽

Primary Bone ◽

Number Of Cells

Abstract Background: Osteosarcoma (OS) is the most common primary bone tumor in both humans and canines. This tumor has an aggressive course leading to the development of metastatic lesions in most patients diagnosed with this disease. Two new novel agents, MLN9708 and SH4-54, work as a proteasome inhibitor and a STAT3 inhibitor, respectively. Targets of these drugs have been shown to be overexpressed in OS in both species. Methods: Two human and two canine OS cell lines were exposed in vitro to both drugs alone and in combination. The number of cells undergoing apoptosis, as well as the number of cells capable of invasion through a matrigel basement membrane was evaluated after exposure to the drugs. Additionally, PCR and Western blots of downstream targets were evaluated. Finally, both drugs were tested against each cell line in an in vivo murine xenograft model. Results: All four cell lines were sensitive to MLN9708, one of the human cell lines and both canine cell lines were resistant to SH4-54. MLN9708 was also better at inhibiting invasion in three of the four cell lines. In the murine xenografts MLN9708 inhibited growth and metastasis in 143B (human OS) and the combination inhibited growth in the canine OS cell line (MCKOS). Conclusions: Though SH4-54 demonstrated robust cell killing in 143B in vitro, MLN9708 demonstrated broader activity across species for the treatment of OS. Further investigation into this drug is warranted as a treatment for OS. Combination of this drug with a STAT3 inhibitor may be worthwhile in canine OS.

Download Full-text

Single-Domain Antibodies for Targeting, Detection, and In Vivo Imaging of Human CD4+ Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.799910 ◽

2021 ◽

Vol 12 ◽

Author(s):

Bjoern Traenkle ◽

Philipp D. Kaiser ◽

Stefania Pezzana ◽

Jennifer Richardson ◽

Marius Gramlich ◽

...

Keyword(s):

T Cell ◽

Immune Cell ◽

Inflammatory Diseases ◽

Xenograft Model ◽

Single Domain ◽

Positron Emission ◽

Depth Analysis ◽

Single Domain Antibodies

The advancement of new immunotherapies necessitates appropriate probes to monitor the presence and distribution of distinct immune cell populations. Considering the key role of CD4+ cells in regulating immunological processes, we generated novel single-domain antibodies [nanobodies (Nbs)] that specifically recognize human CD4. After in-depth analysis of their binding properties, recognized epitopes, and effects on T-cell proliferation, activation, and cytokine release, we selected CD4-specific Nbs that did not interfere with crucial T-cell processes in vitro and converted them into immune tracers for noninvasive molecular imaging. By optical imaging, we demonstrated the ability of a high-affinity CD4-Nb to specifically visualize CD4+ cells in vivo using a xenograft model. Furthermore, quantitative high-resolution immune positron emission tomography (immunoPET)/MR of a human CD4 knock-in mouse model showed rapid accumulation of 64Cu-radiolabeled CD4-Nb1 in CD4+ T cell-rich tissues. We propose that the CD4-Nbs presented here could serve as versatile probes for stratifying patients and monitoring individual immune responses during personalized immunotherapy in both cancer and inflammatory diseases.

Download Full-text

An optimal anti-tumor response by a novel CEA/CD3 bispecific antibody for colorectal cancers

Antibody Therapeutics ◽

10.1093/abt/tbab009 ◽

2021 ◽

Author(s):

Ninghai Wang ◽

Harshal Patel ◽

Irene Schneider ◽

Xin Kai ◽

Avanish K Varshney ◽

...

Keyword(s):

T Cell ◽

Binding Affinity ◽

Tumor Antigen ◽

Xenograft Model ◽

Variable Region ◽

Dependent Manner ◽

Cytokine Release ◽

Complex Development

Abstract Background CD3-based bispecific T cell engagers (bsTCEs) are one of the most promising bispecific antibodies for effective cancer treatments. To elicit target-specific T cell-mediated cytotoxicity, these bsTCEs contain at least one binding unit directed against a tumor antigen and another binding unit targeting CD3 in T cell antigen receptor complex. Development of CD3-based bsTCEs, however, has been severely hampered by dose limiting toxicities due to cytokine release syndrome. To address this limitation, we developed a novel functionally trivalent TCE (t-TCE) antibody containing affinity reduced CD3 binding unit, positioned to ensure monovalent CD3 engagement, in combination with bivalent tumor antigen binding of Carcinoembryonic Antigen (CEA). Methods We modeled the variable region of anti-CD3 in the CDRs of the heavy chain and obtained CD3 binders with reduced binding affinity. Two optimized versions CEA/CD3-v1 and CEA/CD3-v2 were identified and generated in tetravalent format, characterized and compared in vitro and in vivo. Results Our lead candidate, CEA/CD3-v2, demonstrated sub-nanomolar binding and picomolar potency against a panel of CEA-expressing cancer cell lines. In addition, we detected reduced T cell cytokine release with potent cytotoxic activity. Our t-TCE CEA/CD3-v2 molecule demonstrated strong anti-tumor effect in a dose dependent manner in human PBMC xenograft model. Furthermore, combination of CEA/CD3-v2 with atezolizumab provided synergistic antitumor effect. Conclusions Because of effective tumor cell killing with various level of CEA expression and reduced cytokine release, CEA/CD3 BsTCE may greatly benefit in CEA positive cancer immunotherapy. Statement of Significance. Through optimization of CD3 binding affinity and tetravalent format with functional monovalent binding to CD3, t-TCE CEA/CD3–2 molecule not only retains high potency in vitro and in vivo, but also significantly reduces cytokine release.

Download Full-text

TRIB3 promotes hepatocellular carcinoma growth and predicts poor prognosis

Cancer Biomarkers ◽

10.3233/cbm-201577 ◽

2020 ◽

Vol 29 (3) ◽

pp. 307-315

Author(s):

Xiao-Jun Wang ◽

Fei-Fei Li ◽

Yi-Jing Zhang ◽

Man Jiang ◽

Wan-Hua Ren

Keyword(s):

Hepatocellular Carcinoma ◽

Poor Prognosis ◽

Xenograft Model ◽

Clinicopathological Characteristics ◽

Tissue Samples ◽

Normal Tissues ◽

Related Family ◽

Hcc Cells

BACKGROUND: Tribbles pseudokinase 3 (TRIB3) is a member of the tribbles-related family, which is involved a lot of cellular processes and multiple cancers, such as breast cancer, colorectal cancer, renal cell carcinomas, and lung cancer. However, the expression pattern and biological function of TRIB3 in hepatocellular carcinoma (HCC) has not yet been completely elucidated. METHODS: The expression of TRIB3 and clinicopathological characteristics were evaluated by HCC tissue microarray and qPCR analysis. Lentivirus packaging and transfection were employed to establish cell lines with TRIB3 overexpression or knockdown. The biological functions of TRIB3 in the growth of HCC were determined using MTT and crystal violet assays. Tumor growth was monitored in a xenograft model in vivo. RESULTS: The expression of TRIB3 was upregulated in HCC tissue samples compared to paired normal tissues in 45 patients examined by qPCR assay. TRIB3 expression was significantly correlated with HCC tumor size and prognosis in postoperative patients by analysis of the TRIB3 expression data and HCC clinical features. Forced expression of TRIB3 significantly promoted HCC growth in vitro. In contrast, downregulation of TRIB3 inhibited cell growth in vitro. Moreover, knockdown of TRIB3 suppressed tumorigenesis of HCC cells in vivo. CONCLUSION: TRIB3 promotes growth abilities of HCC cells both in vitro and in vivo and predicts poor prognosis of HCC patients, which serves as a prognostic marker and might provide a potential therapeutic candidate for patients with HCC.

Download Full-text

Interrogating resistance mechanisms to PD-1 blockade therapy with CRISPR.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.3077 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 3077-3077

Author(s):

Davis Yuri Torrejon ◽

Jesse Meir Zaretsky ◽

Daniel Sanghoon Shin ◽

Mykola Onyshchenko ◽

Gabriel Abril-Rodriguez ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Cell Line ◽

Wild Type ◽

Antitumor Effects ◽

Ifn Gamma ◽

Syngeneic Mouse Model

3077 Background: We tested the biological significance of the loss of function (LOF) mutations in JAK1 or JAK2 within the IFN-receptor-pathway and in beta-2-microglobulin (B2M), which had been found in patient biopsies with resistance to anti-PD-1 therapy. Methods: We used CRISPR/Cas9 genome editing to generate JAK1, JAK2 and B2M knockout (KO) sublines of HLA-A*02:01 MART-1 or NY-ESO-1 positive human melanoma cell lines, tested using in-vitro T cell co-culture systems and in a syngeneic mouse model (MC38) to analyze the in-vivo antitumor activity with anti-PD1 therapy. Results: The JAK2-KO cell line was insensitive to IFN-gamma induced signaling and growth arrest (p < 0.001 compared with IFN-alpha or beta), while the JAK1-KO cell line was insensitive to all three IFNs. Baseline MHC class I expression after JAK1-KO was unaffected (baseline-MFI 1230 JAK1-KO vs 1570 parental, p = 0.66), but the magnitude of change was lower upon IFN-gamma exposure compared to the parental (MFI change with IFN-gamma, 26% decrease for JAK1-KO vs 50% increase for parental). There was no difference in in-vitro cytotoxicity by NY-ESO-1-TCR transgenic T-cells against JAK1-KO-NY-ESO-1+ melanoma cells compared to the parental (78% vs 82% cytotoxicity at 10:1 E:T ratio, p NS). However, B2M-KO was resistant to killing by MART-1 specific T-cells (2% vs 96% cytotoxicity at 10:1 E:T ratio, p < 0.0001). On the other hand, in the MC38 model the significant antitumor activity of anti-PD-1 against the wild type cells was lost in both JAK2-KO and B2M-KO. The percentage of CD8+ T cells has a trend of increase with anti-PD1 compared to untreated in the MC38 wild type (p = 0.1 d12), and a trend of decrease in MC38 B2M-KO (p = 0.2 d12), but no change in JAK2-KO tumors (p = 0.7 d12). Conclusions: JAK1/2 LOF mutations result in insensitivity to IFN induced antitumor effects, but does not impair T cell recognition and cytotoxicity, while B2M LOF results in lack of antigen presentation to T cells and loss of antitumor activity. However both lead to in-vivo resistance to anti-PD-1 therapy, suggesting they do so by independent mechanisms.

Download Full-text